Andrews N W, Colli W
J Protozool. 1982 May;29(2):264-9. doi: 10.1111/j.1550-7408.1982.tb04024.x.
A quantitative method for experimentally separating the adhesion and interiorization phases of the interaction of Trypanosoma cruzi with mammalian cells was developed. Incubation of confluent monolayers of mammalian cells with epimastigotes or trypomastigotes at 4 degrees C allowed the evaluation of the number of adhered parasites that do not become interiorized at this temperature. Quantification of interiorized parasites at 34 degrees C was achieved by employing hypotonic lysis to eliminate the extracellularly adhered trypomastigotes. Both adhesion and interiorization were found to be proportional to the time of exposure of cells to parasites and to the multiplicity of infection. These phenomena occur normally for trypomastigotes in the absence of serum with LLC-MK2 cells, HeLa cells, and 3T3 fibroblasts. Moreover, it was possible to obtain trypomastigotes that presented the same infectivity to LLC-MK2 cells as did parasites obtained in the presence of 2% fetal calf serum after 10 serial passages in a medium devoid of serum. Inhibition of adhesion (of epimastigotes and trypomastigotes) and of interiorization (of trypomastigotes) was obtained with inactivated normal serum from several sources, a saturation effect being observed at a final concentration of 20%. Bovine serum albumin, at the concentrations present in the sera, had no inhibitory effect. Trypomastigotes that have been pre-incubated with 40% FCS (45 min at 4 degrees C) showed decreased adhesion and interiorization indices, effects that can be reversed by trypsinization of the parasites prior to exposure of the cells. A progressive internalization of previously attached trypomastigotes was observed on raising the temperature from 4 degrees C to 34 degrees C; no spontaneous detachment of parasites was detected up to 120 min. Approximately 75% of the adhered parasites were found inside the cells after 45 min at 34 degrees C. The presence of normal inactivated calf serum during incubation at 34 degrees C resulted in a certain degree of detachment and in a lower interiorization index.
开发了一种定量方法，用于通过实验分离克氏锥虫与哺乳动物细胞相互作用的黏附阶段和内化阶段。将哺乳动物细胞的汇合单层与无鞭毛体或 trypomastigotes 在 4℃ 下孵育，可以评估在此温度下未内化的黏附寄生虫数量。通过采用低渗裂解来消除细胞外黏附的 trypomastigotes，实现了在 34℃ 下对内化寄生虫的定量。发现黏附和内化均与细胞暴露于寄生虫的时间以及感染复数成正比。在无血清条件下，trypomastigotes 与 LLC-MK2 细胞、HeLa 细胞和 3T3 成纤维细胞正常发生这些现象。此外，在无血清培养基中连续传代 10 次后，有可能获得对 LLC-MK2 细胞具有与在 2% 胎牛血清存在下获得的寄生虫相同感染性的 trypomastigotes。用几种来源的灭活正常血清可抑制（无鞭毛体和 trypomastigotes 的）黏附和（trypomastigotes 的）内化，在终浓度为 20% 时观察到饱和效应。血清中存在的浓度的牛血清白蛋白没有抑制作用。预先在 40% FCS 中孵育（4℃ 下 45 分钟）的 trypomastigotes 显示黏附和内化指数降低，在细胞暴露之前对寄生虫进行胰蛋白酶消化可逆转这些效应。将温度从 4℃ 升高到 34℃ 时，观察到先前附着的 trypomastigotes 逐渐内化；在长达 120 分钟内未检测到寄生虫的自发脱离。在 34℃ 孵育期间存在正常灭活小牛血清会导致一定程度的脱离和较低的内化指数。
